肠出血性大肠杆菌转移紧密黏附素受体的表达及其活性鉴定  

Prokaryotic Expression and Biological Activity Identification of Tir of Enterohemorrhagic Escherichia coli

作　　者：肖乐[1] 王琴[1] 蔡昆[1] 屠伟[1] 侯晓军[1] 李涛[1] 王慧[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《现代生物医学进展》2010年第9期1646-1649,共4页Progress in Modern Biomedicine

摘　　要：目的:在原核表达基因工程菌中实现肠出血性大肠杆菌(EHEC)O157:H7转移紧密黏附素受体(Translocated Intim in Receptor,Tir)蛋白的高效表达,并对其活性进行初步鉴定。方法:采用PCR方法从EHEC O157:H7基因组中调取tir基因,插入pEASY-T1克隆载体。克隆质粒测序鉴定后,采用NdeⅠ、XhoⅠ限制性核酸内切酶双酶切pEASY-T1-tir质粒获得tir基因,连接同样经过双酶切的pET-22b(+)。表达质粒转化E.coliBL21(DE3),IPTG诱导表达,SDS-PAGE检测相对分子质量,Western blotting验证抗原活性。荧光显微镜观察蛋白是否具有嵌入细胞膜的活性。结果:PCR扩增得到1686bp的目的片段。构建的原核表达质粒pET-22b(+)-tir经酶切鉴定及测序与预期序列一致。目的蛋白以裂解上清形式表达,表达量约3mg/ml。经镍柱纯化后纯度达90%以上。重组表达的Tir具有嵌入细胞膜的生物学功能。结论:成功表达了具有生物活性的重组Tir蛋白,为Tir的功能研究奠定基础。Objectives：To express full-length Tir of enterohemorrhagic Escherichia coli（EHEC） in E.coli BL21 and to identify the biologcal activity of Tir.Methods：The tir gene was cloned from EHEC O157：H7 EDL 933 by PCR.The production of PCR was inserted into pEASY-T1 Simple cloning vector.The gene tir in cloning plasmid was sequenced,and was cut down by NdeⅠand XhoⅠendonucleases.Then it was linked with pET-22b（＋）.The expression plasmid was transformed into E.coli BL21（DE3）,and induced by IPTG to express the recombinant Tir.The Tir protein was detected by SDS-PAGE,and the conformation was validated by Western blotting.The biological activity was identified by fluorescence microscope.Results：The 1686bp DNA was gained.The plasmid pET-22b（＋）-tir was builded.Tir was expressed in supernatant of lysis and the protein was purified by Ni2＋ affinity chromatograghy.The recombinant protein Tir was capable of inserting the membrane of Hep2 cell.Conclusion：The recombinant Tir was expressed,successfully,with biological activity,which deserves further investigations for its functions.

关 键 词：肠出血性大肠杆菌 TIR 表达 活性鉴定 

分 类 号：Q78[生物学—分子生物学] Q75